Chapter 21 Quantification of Murine AAT by Direct ELISA Andrew Cox and Christian Mueller Abstract This methods chapter elaborates on how a direct enzyme-linked immunosorbent assay (ELISA) is used to specifically detect and quantify murine alpha-1 antitrypsin (AAT). As a direct ELISA, it lacks some sensitivity as compared to the “sandwich” ELISA method; however, it does reliably differentiate between samples with varying amounts of the mouse AAT protein. This protocol relies on the principle of adsorption to coat each well with sera proteins, whereas detection occurs specifically using a two-step antibody combination. This procedure effectively identifies and quantifies murine AAT from a wide variety of samples including mouse serum, cell culture medium, and cell or tissue lysate. Key words Alpha-1 antitrypsin, AAT, Murine AAT, Enzyme-linked immunosorbent assay, Direct ELISA
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Introduction The enzyme-linked immunosorbent assay (ELISA) is a quantitative protein plate-based assay analogous to immunodetection of proteins using a membrane [1]. More specifically, the ELISA quantifies a protein of interest that is in solution [2]. The direct ELISA, as compared to the “sandwich” ELISA, relies on the immobilization of a protein directly to the polystyrene well rather than coating with an initial capture antibody [3]. In this regard, the direct ELISA can be less sensitive as all sample components can bind with the well rather than just the protein of interest that will bind to the capture antibody [2]. In this protocol, two antibodies are used. The primary binds a specific epitope of the protein of interest, while the secondary (conjugated with a reporter) detects the primary [1]. After both interactions occur, the collective signal strength of the reporter is detected and quantified. Extrapolation from a standard curve and accounting for the sample dilution renders a quantifiable protein reading from the sample of interest. This chapter outlines how to perform the murine AAT direct ELISA. As such, this protocol allowed for the identification and
Florie Borel and Christian Mueller (eds.), Alpha-1 Antitrypsin Deficiency: Methods and Protocols, Methods in Molecular Biology, vol. 1639, DOI 10.1007/978-1-4939-7163-3_21, © Springer Science+Business Media LLC 2017
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differentiation of homozygous, heterozygous, and knockout mice that were bred in an AAT knockout study. Alpha-1 antitrypsin, as a secreted serpin protein, is naturally prevalent in sera samples, making it convenient to identify and quantify using the ELISA method. It is also possible to perform the same ELISA using cell culture media and tissue or cell lysates; however, expect to troubleshoot different sample dilutions [2].
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Materials Prepare and store all reagents at room temperature (unless otherwise indicated). Materials for waste disposal are to adhere to the institutional waste disposal regulations. All solutions are to be prepared using ultrapure water. 1. Clear flat-bottom 96-well microtiter plate. 2. Prepare 0.2 M sodium carbonate–bicarbonate coating buffer and adjust pH to 9.4 with NaOH. Autoclave and store at room temperature. 3. Prepare mouse AAT reference standard (see Note 1). Dilute mouse alpha-1 antitrypsin to 1000 ng/mL (Cat# RS-90A1T, Immunology Consultants Laboratory Inc., Portland, OR) in coating buffer. Store stock in 10 μL aliquots at 20 C. Serially dilute to 500 ng/mL 1:2 (include 250 ng/mL, 125 ng/mL, 62.5 ng/mL, 31.25 ng/mL, 15.63 ng/mL, 7.81 ng/mL, and 3.9 ng/mL and 0 ng/mL). 4. Microplate sealing films. 5. Prepare 1 PBS-0.05% Tween 20 washing solution. 6. Prepare 2% skim milk blocking solution in 1 PBS–0.05% Tween. 7. Prepare unknown samples and controls (see Notes 1 and 2). Negative control as human serum (pooled normal human serum, Innovative Research and Technology, Cat# 1PLA-1). Make 50 μL aliquots and store at 80 C. Positive control: serum from C57BL/6 mouse. 8. Prepare 1:2000 primary antibody (goat anti-mouse Alpha-1 Antitrypsin, Cat# GA1T-90A-Z, Immunology Consultants Laboratory Inc) in 2% skim milk blocking solution via 2-step dilution. Store stock solution at 4 C. 9. Prepare 1:10,000 secondary detection antibody (HRPconjugated rabbit anti-goat IgG h+l). Store stock at 4 C. 10. Prepare freshly the TMB peroxidase substrate (TMB microwell peroxidase substrate system, Cat# 50-76-03, KPL Scientific, Gaithersburg, MD): mix equal volumes (5.5 mL each) of TMB
Direct Murine AAT ELISA
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Peroxidase Substrate and Peroxidase Substrate Solution B. Store stocks at 4 C. 11. Prepare 10% stop solution: 22.2 mL of 95–98% sulfuric acid, 377.8 mL of water. 12. Multichannel pipette. 13. Laboratory vacuum suction system. 14. Dilution plates. 15. Plate washer (optional), such as MultiWasher III (Cat# 844107, TriContinent, Grass Valley, CA). 16. Microplate reader with 450 nm filter, such as VersaMax (Molecular Devices, Sunnyvale, CA). 17. Software (optional), such as Softmax Pro (Molecular Devices).
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Methods 1. Coat plate with 100 μL each of appropriately diluted standards (Table 1), blank (coating buffer), unknown samples, and controls (Table 2) to assigned wells (see Notes 1 and 2). 2. Cover microplate with film and incubate for 1 h at 37 C (see Note 3).
Table 1 Murine AAT standards are prepared according to chart above. 1000 ng/mL starting solution is prepared using a two-step initial dilution
Step
Final AAT concentration (ng/mL) AAT standard
Sample diluent (coating buffer) (μL)
2 step: 0
(a) 10,000 (b) 1000
1 μL 2.44 mg/mL stock 50 μL from step 0a
243 450
1
500
250 μL from step 0b
250
2
250
250 μL from step 1
250
3
125
250 μL from step 2
250
4
62.5
250 μL from step 3
250
5
31.25
250 μL from step 4
250
6
15.625
250 μL from step 5
250
7
7.8125
250 μL from step 6
250
8
3.90625
250 μL from step 7
250
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Andrew Cox and Christian Mueller
Table 2 +/ controls and unknown sample dilution are prepared using this twostep 1:2500 dilution scale. Controls and unknown samples to fall within the concentration range of standards
Step
Final dilution
Sample
Sample diluent (coating buffer) (μL)
1
1:100
2μL of stock
198
2
1:2500
10μL from step 1
240
3. Wash plate 4 with 300 μL PBS–Tween 20 washing solution. 4. Aspirate remaining liquid in wells. 5. Add 100 μL of 2% skim milk 1PBS–Tween 20 blocking solution to wells. 6. Cover with film and incubate for 1 h at 37 C (see Note 3). 7. Wash plate 4 with 300 μL PBS–Tween 20 washing solution. 8. Aspirate remaining liquid in wells. 9. Add 100 μL of 1:2000 primary antibody. 10. Cover with film and incubate at 37 C. 11. Wash plate 4 with 300 μL of PBS–Tween 20 washing solution. 12. Aspirate remaining liquid in wells. 13. Add 100μL of 1:10,000 secondary HRP conjugated detection antibody. 14. Cover with film and incubate at 37 C. 15. Wash plate 4 with 300 μL PBS–Tween 20 washing solution. 16. Aspirate remaining liquid in wells. 17. Add 100 μL of mixed 1:1 TMB peroxidase substrate. 18. Incubate for 10–15 min at room temperature. 19. Add 50 μL of 10% stop solution. 20. Read microplate at OD of 450 nm. 21. Fit standard curve as trend line (4-parameter or linear regression) and calculate R2 (see Note 4). 22. Use trend line fit curve to calculate the concentration of unknown samples (Figs. 1 and 2)
Direct Murine AAT ELISA
221
Fig. 1 Demonstrates an example of varying AAT levels in sera samples from male homozygous, heterozygous, and knockout mice. Positive Control: serum from male C57BL6 mouse, negative control: human serum. Data extrapolated from semilog linear standard
Fig. 2 Demonstrates an example of varying AAT levels in sera samples from female homozygous, heterozygous, and knockout mice. Positive Control: serum from female C57BL6 mouse, negative control: human serum. Data extrapolated from semilog linear standard
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Notes 1. It is highly desirable to run standards and unknown samples in duplicate or triplicate. 2. Depending on the samples’ concentration of AAT, dilutions may be necessary. Use coating buffer as diluting agent. Dilution has to be determined experimentally. 3. One hour-room temperature steps in this protocol can be substituted by incubating overnight at 4 C. 4. R2 with a 4-parameter fit should be 0.99. If using linear fit, use semilog scaling option, recommend R2 0.97. Only interpret points falling within range of standard. Expand standard or increase dilution accordingly.
References 1. Qiagen Bench Guide (2001) Protein Assay, 83–87 2. Ausubel F et al (2003) Current protocols in molecular biology. John Wiley & Sons, New York. (pages 1661, Section 11.2.1-22)
3. KPL Technical Guide for ELISA (2013) Assay Formats, 5–7
1
Introduction The enzyme-linked immunosorbent assay (ELISA) is a quantitative protein plate-based assay analogous to immunodetection of proteins using a membrane [1]. More specifically, the ELISA quantifies a protein of interest that is in solution [2]. The direct ELISA, as compared to the “sandwich” ELISA, relies on the immobilization of a protein directly to the polystyrene well rather than coating with an initial capture antibody [3]. In this regard, the direct ELISA can be less sensitive as all sample components can bind with the well rather than just the protein of interest that will bind to the capture antibody [2]. In this protocol, two antibodies are used. The primary binds a specific epitope of the protein of interest, while the secondary (conjugated with a reporter) detects the primary [1]. After both interactions occur, the collective signal strength of the reporter is detected and quantified. Extrapolation from a standard curve and accounting for the sample dilution renders a quantifiable protein reading from the sample of interest. This chapter outlines how to perform the murine AAT direct ELISA. As such, this protocol allowed for the identification and
Florie Borel and Christian Mueller (eds.), Alpha-1 Antitrypsin Deficiency: Methods and Protocols, Methods in Molecular Biology, vol. 1639, DOI 10.1007/978-1-4939-7163-3_21, © Springer Science+Business Media LLC 2017
217
218
Andrew Cox and Christian Mueller
differentiation of homozygous, heterozygous, and knockout mice that were bred in an AAT knockout study. Alpha-1 antitrypsin, as a secreted serpin protein, is naturally prevalent in sera samples, making it convenient to identify and quantify using the ELISA method. It is also possible to perform the same ELISA using cell culture media and tissue or cell lysates; however, expect to troubleshoot different sample dilutions [2].
2
Materials Prepare and store all reagents at room temperature (unless otherwise indicated). Materials for waste disposal are to adhere to the institutional waste disposal regulations. All solutions are to be prepared using ultrapure water. 1. Clear flat-bottom 96-well microtiter plate. 2. Prepare 0.2 M sodium carbonate–bicarbonate coating buffer and adjust pH to 9.4 with NaOH. Autoclave and store at room temperature. 3. Prepare mouse AAT reference standard (see Note 1). Dilute mouse alpha-1 antitrypsin to 1000 ng/mL (Cat# RS-90A1T, Immunology Consultants Laboratory Inc., Portland, OR) in coating buffer. Store stock in 10 μL aliquots at 20 C. Serially dilute to 500 ng/mL 1:2 (include 250 ng/mL, 125 ng/mL, 62.5 ng/mL, 31.25 ng/mL, 15.63 ng/mL, 7.81 ng/mL, and 3.9 ng/mL and 0 ng/mL). 4. Microplate sealing films. 5. Prepare 1 PBS-0.05% Tween 20 washing solution. 6. Prepare 2% skim milk blocking solution in 1 PBS–0.05% Tween. 7. Prepare unknown samples and controls (see Notes 1 and 2). Negative control as human serum (pooled normal human serum, Innovative Research and Technology, Cat# 1PLA-1). Make 50 μL aliquots and store at 80 C. Positive control: serum from C57BL/6 mouse. 8. Prepare 1:2000 primary antibody (goat anti-mouse Alpha-1 Antitrypsin, Cat# GA1T-90A-Z, Immunology Consultants Laboratory Inc) in 2% skim milk blocking solution via 2-step dilution. Store stock solution at 4 C. 9. Prepare 1:10,000 secondary detection antibody (HRPconjugated rabbit anti-goat IgG h+l). Store stock at 4 C. 10. Prepare freshly the TMB peroxidase substrate (TMB microwell peroxidase substrate system, Cat# 50-76-03, KPL Scientific, Gaithersburg, MD): mix equal volumes (5.5 mL each) of TMB
Direct Murine AAT ELISA
219
Peroxidase Substrate and Peroxidase Substrate Solution B. Store stocks at 4 C. 11. Prepare 10% stop solution: 22.2 mL of 95–98% sulfuric acid, 377.8 mL of water. 12. Multichannel pipette. 13. Laboratory vacuum suction system. 14. Dilution plates. 15. Plate washer (optional), such as MultiWasher III (Cat# 844107, TriContinent, Grass Valley, CA). 16. Microplate reader with 450 nm filter, such as VersaMax (Molecular Devices, Sunnyvale, CA). 17. Software (optional), such as Softmax Pro (Molecular Devices).
3
Methods 1. Coat plate with 100 μL each of appropriately diluted standards (Table 1), blank (coating buffer), unknown samples, and controls (Table 2) to assigned wells (see Notes 1 and 2). 2. Cover microplate with film and incubate for 1 h at 37 C (see Note 3).
Table 1 Murine AAT standards are prepared according to chart above. 1000 ng/mL starting solution is prepared using a two-step initial dilution
Step
Final AAT concentration (ng/mL) AAT standard
Sample diluent (coating buffer) (μL)
2 step: 0
(a) 10,000 (b) 1000
1 μL 2.44 mg/mL stock 50 μL from step 0a
243 450
1
500
250 μL from step 0b
250
2
250
250 μL from step 1
250
3
125
250 μL from step 2
250
4
62.5
250 μL from step 3
250
5
31.25
250 μL from step 4
250
6
15.625
250 μL from step 5
250
7
7.8125
250 μL from step 6
250
8
3.90625
250 μL from step 7
250
220
Andrew Cox and Christian Mueller
Table 2 +/ controls and unknown sample dilution are prepared using this twostep 1:2500 dilution scale. Controls and unknown samples to fall within the concentration range of standards
Step
Final dilution
Sample
Sample diluent (coating buffer) (μL)
1
1:100
2μL of stock
198
2
1:2500
10μL from step 1
240
3. Wash plate 4 with 300 μL PBS–Tween 20 washing solution. 4. Aspirate remaining liquid in wells. 5. Add 100 μL of 2% skim milk 1PBS–Tween 20 blocking solution to wells. 6. Cover with film and incubate for 1 h at 37 C (see Note 3). 7. Wash plate 4 with 300 μL PBS–Tween 20 washing solution. 8. Aspirate remaining liquid in wells. 9. Add 100 μL of 1:2000 primary antibody. 10. Cover with film and incubate at 37 C. 11. Wash plate 4 with 300 μL of PBS–Tween 20 washing solution. 12. Aspirate remaining liquid in wells. 13. Add 100μL of 1:10,000 secondary HRP conjugated detection antibody. 14. Cover with film and incubate at 37 C. 15. Wash plate 4 with 300 μL PBS–Tween 20 washing solution. 16. Aspirate remaining liquid in wells. 17. Add 100 μL of mixed 1:1 TMB peroxidase substrate. 18. Incubate for 10–15 min at room temperature. 19. Add 50 μL of 10% stop solution. 20. Read microplate at OD of 450 nm. 21. Fit standard curve as trend line (4-parameter or linear regression) and calculate R2 (see Note 4). 22. Use trend line fit curve to calculate the concentration of unknown samples (Figs. 1 and 2)
Direct Murine AAT ELISA
221
Fig. 1 Demonstrates an example of varying AAT levels in sera samples from male homozygous, heterozygous, and knockout mice. Positive Control: serum from male C57BL6 mouse, negative control: human serum. Data extrapolated from semilog linear standard
Fig. 2 Demonstrates an example of varying AAT levels in sera samples from female homozygous, heterozygous, and knockout mice. Positive Control: serum from female C57BL6 mouse, negative control: human serum. Data extrapolated from semilog linear standard
222
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Andrew Cox and Christian Mueller
Notes 1. It is highly desirable to run standards and unknown samples in duplicate or triplicate. 2. Depending on the samples’ concentration of AAT, dilutions may be necessary. Use coating buffer as diluting agent. Dilution has to be determined experimentally. 3. One hour-room temperature steps in this protocol can be substituted by incubating overnight at 4 C. 4. R2 with a 4-parameter fit should be 0.99. If using linear fit, use semilog scaling option, recommend R2 0.97. Only interpret points falling within range of standard. Expand standard or increase dilution accordingly.
References 1. Qiagen Bench Guide (2001) Protein Assay, 83–87 2. Ausubel F et al (2003) Current protocols in molecular biology. John Wiley & Sons, New York. (pages 1661, Section 11.2.1-22)
3. KPL Technical Guide for ELISA (2013) Assay Formats, 5–7
